Hepatitis C virus asialoglycoproteins

ABSTRACT

Two Hepatitis C Virus envelope proteins (E1 and E2) are expressed without sialylation. Recombinant expression of these proteins in lower eukaryotes, or in mammalian cells in which terminal glycosylation is blocked, results in recombinant proteins which are more similar to native HCV glycoproteins. When isolated by GNA lectin affinity, the E1 and E2 proteins aggregate into virus-like particles.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/249,843,filed May 26, 1994, which is a continuation-in-part of U.S. Ser. No.07/758,880, filed Sep. 13, 1991, now abandoned which is acontinuation-in-part of U.S. Ser. No. 07/611,419, filed Nov. 8, 1990,now abandoned, the discosures of which are incorporated herein byreference.

DESCRIPTION

1. Technical Field

This invention relates to the general fields of recombinant proteinexpression and virology. More particularly, the invention relates toglycoproteins useful for diagnosis, treatment, and prophylaxis ofHepatitis C virus (HCV) infection, and methods for producing suchglycoproteins.

2. Background of the Invention

Non-A, Non-B hepatitis (NANBH) is a transmissible disease (or family ofdiseases) that is believed to be virtually induced, and isdistinguishable from other forms of virus-associated liver disease, suchas those caused by hepatitis A virus (HAV), hepatitis B virus (HBV),delta hepatitis virus (HDV), cytomegalovirus (CMV) or Epstein-Barr virus(EBV). Epidemiologic evidence suggests that there may be three types ofNANBH: the water-borne epidemic type; the blood or needle associatedtype; and the sporadically occurring community acquired type. The numberof causative agents is unknown. However, a new viral species, hepatitisC virus (HCV) has recently been identified as the primary (if not only)cause of blood-borne NANBH (BB-NANBH). See for example PCT WO89/046699and U.S. patent application Ser. No. 07/355,002, filed May 18, 1989.Hepatitis C appears to be the major form of transfusion-associatedhepatitis in a number of countries or regions, including the UnitedStates, Europe, and Japan. There is also evidence implicating HCV ininduction of hepatocellular carcinoma. Thus, a need exists for aneffective method for preventing and treating HCV infection.

The demand for sensitive, specific methods for screening and identifyingcarriers of HCV and HCV-contaminated blood or blood products issignificant. Post-transfusion hepatitis (PITH) occurs in approximately10% of transfused patients, and HCV accounts for up to 90% of thesecases. The major problem in this disease is the frequent progression tochronic liver damage (25-55%).

Patient care, as well as the prevention of transmission of HCV by bloodand blood products or by close personal contact, requires reliablediagnostic and prognostic tools to detect nucleic acids, antigens andantibodies related to HCV. In addition, there is also a need foreffective vaccines and immunotherapeutic therapeutic agents for theprevention and/or treatment of the disease.

HCV appears in the blood of infected individuals at very low ratesrelative to other infectious viruses, which makes the virus verydifficult to detect. The low viral burden is probably the primary reasonthat the causative agent of NANB hepatitis went so long undetected. Eventhough it has now been cloned, HCV still proves difficult to culture andpropagate. Accordingly, there is a strong need for recombinant means ofproducing diagnostic/therapeutic/prophylactic HCV proteins.

3. Disclosure of the Invention

It has been found that two HCV proteins, E1 and E2, appear to bemembrane associated asialoglycoproteins when expressed in recombinantsystems. This is surprising because glycoproteins do not usually remainin mannose-terminated form in mammals, but are further modified withother carbohydrates: the mannose-terminated form is typically onlytransient. In the case of E1 and E2 (as expressed in our systems), theasialoglycoprotein appears to be the final form. E1 (envelope protein 1)is a glycoprotein having a molecular weight of about 35 kD which istranslated from the predicted E1 region of the HCV genome. E2 (envelopeprotein 2) is a glycoprotein having a molecular weight of about 72 kDwhich is translated from the predicted NS1 (non-structural protein 1)region of the HCV genome, based on the flaviviral model of HCV. As viralglycoproteins are often highly immunogenic, E1 and E2 are primecandidates for use in immuno-assays and therapeutic/prophylacticvaccines.

The discovery that E1 and E2 are not sialylated is significant. Theparticular form of a protein often dictates which cells may serve assuitable hosts for recombinant expression. Prokaryotes such as E. colido not glycosylate proteins, and are generally not suitable forproduction of glycoproteins for use as antigens because glycosylation isoften important for full antigenicity, solubility, and stability of theprotein. Lower eukaryotes such as yeast and fungi glycosylate proteins,but are generally unable to add terminal sialic acid residues to thecarbohydrate complexes. Thus, yeast-derived proteins may beantigenically distinct from their natural (non-recombinant)counterparts. Expression in mammalian cells is preferred forapplications in which the antigenicity of the product is important, asthe glycosylation of the recombinant protein should closely resemblethat of the wild viral proteins.

New evidence indicates that the HCV virus may gain entry to host cellsduring infection through either the asialoglycoprotein receptor found onhepatocytes, or through the mannose receptor found on hepaticendothelial cells and macrophages (particularly Kupffer cells).Surprisingly, it has been found that the bulk of natural E1 and E2 donot contain terminal sialic acid residues, but are onlycore-glycosylated. A small fraction additionally contains terminalN-acetylglucosamine. Accordingly, it is an object of the presentinvention to provide HCV envelope glycoproteins lacking all orsubstantially all terminal sialic acid residues.

Another aspect of the invention is a method for producing asialo-E1 orE2, under conditions inhibiting addition of terminal sialic acid, e.g.,by expression in yeast or by expression in mammalian cells usingantibiotics to facilitate secretion or release.

Another aspect of the invention is a method for purifying E1 or E2 byaffinity to lectins which bind terminal mannose residues or terminalN-acetylglucosamine residues.

Another aspect of the invention is an immunogenic composition comprisinga recombinant asialoglycoprotein selected from the group consisting ofHCV E1 and E2 in combination with a pharmaceutically acceptable vehicle.One may optionally include an immunological adjuvant, if desired.

Another aspect of the invention is an immunoassay reagent, comprising arecombinant asialoglycoprotein selected from the group consisting of HCVE1 and E2 in combination with a suitable support. Another immunoassayreagent of the invention comprises a recombinant asialoglycoproteinselected from the group consisting of HCV E1 and E2 in combination witha suitable detectable label.

Another aspect of the invention concerns dimers and higher-orderaggregates of E1 and/or E2. One species of the invention is an E2complex. Another species of the invention is an E1:E2 heterodimer.

Another aspect of the invention is an HCV vaccine composition comprisingE1:E2 aggregates and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method for purifying E1:E2complexes.

MODES OF CARRYING OUT THE INVENTION A. Definitions

The term "asialoglycoprotein" refers to a glycosylated protein which issubstantially free of sialic acid moieties. Asialoglycoproteins may beprepared recombinantly, or by purification from cell culture or naturalsources. Presently preferred asialoglycoproteins are derived from HCV,preferably the glycoproteins E1 and E2, most preferably recombinant E1and E2 (rE1 and rE2). A protein is "substantially free" of sialic acidwithin the scope of this definition if the amount of sialic acidresidues does not substantially interfere with binding of theglycoprotein to mannose-binding proteins such as GNA. This degree ofsialylation will generally be obtained where less than about 40% of thetotal N-linked carbohydrate is sialic acid, more preferably less thanabout 30%, more preferably less than about 20%, more preferably lessthan about 10%, more preferably less than about 5%, and most preferablyless than about 2%.

The term "E1" as used herein refers to a protein or polypeptideexpressed within the first 400 amino acids of an HCV polyprotein,sometimes referred to as the E or S protein. In its natural form it is a35 kD glycoprotein which is found strongly membrane-associated. In mostnatural HCV strains, the E1 protein is encoded in the viral polyproteinfollowing the C (core) protein. The E1 protein extends fromapproximately amino acid 192 to about aa383 of the full-lengthpolyprotein. The term "E1" as used herein also includes analogs andtruncated mutants which are immunologically crossreactive with naturalE1.

The term "E2" as used herein refers to a protein or polypeptideexpressed within the first 900 amino acids of an HCV polyprotein,sometimes referred to as the NS1 protein. In its natural form it is a 72kD glycoprotein which is found strongly membrane-associated. In mostnatural HCV strains, the E2 protein follows the E1 protein. The E2protein extends from approximately aa384 to about aa820. The term "E2"as used herein also includes analogs and truncated mutants which areimmunologically crossreactive with natural E2.

The term "aggregate" as used herein refers to a complex of E1 and/or E2containing more than one E1 or E2 monomer. E1:E1 dimers, E2:E2 dimers,and E1:E2 heterodimers are all "aggregates" within the scope of thisdefinition. Compositions of the invention may also include largeraggregates, and may have molecular weights in excess of 800 kD.

The term "particle" as used herein refers to an E1, E2, or E1 /E2aggregate visible by electron microscopy and having a dimension of atleast 20 nm. Preferred particles are those having a roughly sphericalappearance and a diameter of approximately 40 nm by electron microscopy.

The term "purified" as applied to proteins herein refers to acomposition wherein the desired protein comprises at least 35% of thetotal protein component in the composition. The desired proteinpreferably comprises at least 40%, more preferably at least about 50%,more preferably at least about 60%, still more preferably at least about70%, even more preferably at least about 80%, even more preferably atleast about 90%, and most preferably at least about 95% of the totalprotein component. The composition may contain other compounds such ascarbohydrates, salts, lipids, solvents, and the like, without affectingthe determination of percentage purity as used herein. An "isolated" HCVasialoglycoprotein intends an HCV asialoglycoprotein composition whichis at least 35% pure.

"Mannose-binding protein" as used herein intends a lectin or otherprotein which specifically binds to proteins having mannose-terminatedglycosylation (e.g., asialoglycoproteins), for example, mannose-bindinglectins, antibodies specific for mannose-terminated glycosylation,mannose receptor protein (R. A. B. Ezekowitz et al., J Exp Med (1990)176:1785-94), asialoglycoprotein receptor proteins (H. Kurata et al., JBiol Chem (1990) 265:11295-98), serum mannose-binding protein (I.Schuffenecker et al., Cytogenet Cell Genet (1991) 56:99-102; K. Sastryet al., J Immunol (1991) 147:692-97), serum asialoglycoprotein-bindingprotein, and the like. Mannose-binding lectins include, for example,GNA, Concanavalin A (ConA), and other lectins with similar bindingproperties.

The term "GNA lectin" refers to Gakinthus nivalus agglutinin, acommercial) available lectin which binds to mannose-terminatedglycoproteins.

A "recombinant" glycoprotein as used herein is a glycoprotein expressedfrom a recombinant polynucleotide, in which the structural gene encodingthe glycoprotein is expressed under the control of regulatory sequencesnot naturally adjacent to the structural gene, or in which thestructural gene is modified. For example, one may form a vector in whichthe E1 structural gene is placed under control of a functional fragmentof the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter.A presently preferred promoter for use in yeast is the hybrid ADH2/GAPpromoter described in U.S. Pat. No. 4,880,734 (incorporated herein byreference), which employs a fragment of the GAPDH promoter incombination with the upstream activation sequence derived from alcoholdehydrogenase 2. Modifications of the structural gene may includesubstitution of different codons with degenerate codons (e.g., toutilize host-preferred codons, eliminate or generate restriction enzymecleavage sites, to control hairpin formation, etc.), and substitution,insertion or deletion of a limited number of codons encoding differentamino acids (preferably no more than about 10%, more preferably lessthan about 5% by number of the natural amino acid sequence should bealtered), and the like. Similarly, a "recombinant" receptor refers to areceptor protein expressed from a recombinant polynucleotide, in whichthe structural gene encoding the receptor is expressed under the controlof regulatory sequences not naturally adjacent to the structural gene,or in which the structural gene is modified.

The term "isolated polypeptide" refers to a polypeptide which issubstantially free of other HCV viral components, particularlypolynucleotides. A polypeptide composition is "substantially free" ofanother component if the weight of the polypeptide in the composition isat least 70% of the weight of the polypeptide and other componentcombined, more preferably at least about 80%, still more preferablyabout 90%, and most preferably 95% or greater. For example, acomposition containing 100 μg/ml E1 and only 3 μg/ml other HCVcomponents (e.g., DNA, lipids, etc.) is substantially free of "other HCVviral components", and thus is a composition of an isolated polypeptidewithin the scope of this definition.

The term "secretion leader" refers to a polypeptide which, when encodedat the N-terminus of a protein, causes the protein to be secreted intothe host cell's culture medium following translation. The secretionleader will generally be derived from the host cell employed. Forexample, suitable secretion leaders for use in yeast include theSaccharomyces cerevisiae α-factor leader (see U.S. Pat. No. 4,870,008,incorporated herein by reference).

The term "lower eukaryote" refers to host cells such as yeast, fungi,and the like. Lower eukaryotes are generally (but not necessarily)unicellular. Preferred lower eukaryotes are yeasts, particularly specieswithin Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia,Hansenula, and the like. Saccharomyces cerevisiae, S. carlsbergensis andK. Lactis are the most commonly used yeast hosts, and are convenientfungal hosts.

The term "higher eukaryote" refers to host cells derived from higheranimals, such as mammals, reptiles, insects, and the like. Presentlypreferred higher eukaryote host cells are derived from Chinese hamster(e.g., CHO), monkey (e.g., COS cells), human, and insect (e.g.,Spodoptera frugiperda). The host cells may be provided in suspension orflask cultures, tissue cultures, organ cultures, and the like.

The term "calcium modulator" refers to a compound capable ofsequestering or binding calcium ions within the endoplasmic reticulum,or affects calcium ion concentration within the ER by its effect oncalcium regulatory proteins (e.g., calcium channel proteins, calciumpumps, etc.). Suitable calcium modulators include, for examplethapsigargin, EGTA (ethylene glycol bis[β-aminoethyl ether]N,N,N',N'-tetraacetic acid). The presently preferred modulator isthapsigargin (see e.g., O. Thastrup et al, Proc Nat Acad Sci USA (1990)87:2466-70).

The term "immunogenic" refers to the ability of a substance to cause ahumoral and/or cellular immune response, whether alone or when linked toa carrier, in the presence or absence of an adjuvant. "Neutralization"refers to an immune response that blocks the infectivity, eitherpartially or fully, of an infectious agent. A "vaccine" is animmunogenic composition capable of eliciting protection against HCV,whether partial or complete, useful for treatment of an individual.

The term "biological liquid" refers to a fluid obtained from anorganism, such as serum, plasma, saliva, gastric secretions, mucus, andthe like. In general, a biological liquid will be screened for thepresence of HCV particles. Some biological fluids are used as a sourceof other products, such as clotting factors (e.g., Factor VIII:C), serumalbumin, growth hormone, and the like. In such cases, it is importantthat the source biological fluid be free of contamination by virus suchas HCV.

B. General Method

The E1 region of the HCV genome is described in EP 388,232 as region"E", while E2 is described as "NS 1." The E1 region comprisesapproximately amino acids 192-383 in the full-length viral polyprotein.The E2 region comprises approximately amino acids 384-820. The completesequences of prototypes of these proteins (strain HCV-1) are availablein the art (see EP 388,232), as are general methods for cloning andexpressing the proteins. Both E1 and E2 may be expressed from apolynucleotide encoding the first 850-900 amino acids of the HCVpolyprotein(see e.g., SEQ. ID NO:3): post-translational processing inmost eukaryotic host cells cleaves the initial polyprotein into C, E1,and E2. One may truncate the 5' end of the coding region to reduce theamount of C protein produced.

Expression of asialoglycoproteins may be achieved by a number ofmethods. For example, one may obtain expression in lower eukaryotes(such as yeast) which do not normally add sialic acid residues toglycosylated proteins. In yeast expression systems, it is presentlypreferred to employ a secretion leader such as the S. cerevisiaeα-factor leader, so that the protein is expressed into the culturemedium following translation. It is also presently preferred to employglycosylation-deficient mutants such as pmr1, as these mutants supplyonly core glycosylation, and often secrete heterologous proteins withhigher efficiency (H. K. Rudolph et al, Cell (1989) 58:133-45).Alternatively, one may employ other species of yeast, such as Pichiapastons, which express glycoproteins containing 8-9 mannose residues ina pattern believed to resemble the core glycosylation pattern observedin mammals and S. cerevisiae.

Alternatively, one may arrange expression in mammalian cells, and blockterminal glycosylation (addition of sialic acid). Recombinant constructswill preferably include a secretion signal to insure that the protein isdirected toward the endoplasmic reticulum. Transport to the golgiappears to be blocked by E1 and E2 themselves: high-level expression ofE1 or E2 in mammalian cells appears to arrest secretion of all cellularproteins at the endoplasmic reticulum or cis golgi. One may additionallyemploy a glycosylation defective mutant. See for example, P. Stanley,Ann Rev Genet (1984) 18:525-52. In the event a glycosylation ortransport mutant expresses E1 or E2 with sialylation, the terminalsialic acid residues may be removed by treatment with neuraminidase.

Yield should be further increased by use of a calcium modulator toobtain release of protein from within the endoplasmic reticulum.Suitable modulators include thapsigargin, EGTA, and A23817 (see e.g., O.Thastrup et al, Proc Nat Acad Sci USA (1990) 87:2466-70). For example,one may express a large amount of E1 or E2 intracellularly in mammaliancells (e.g., CHO, COS, HeLa cells, and the like) by transfection with arecombinant vaccinia virus vector. After allowing time for proteinexpression and accumulation in the endoplasmic reticulum, the cells areexposed to a calcium modulator in concentration large enough to causerelease of the ER contents. The protein is then recovered from theculture medium, which is replaced for the next cycle.

Additionally, it may be advantageous to express a truncated form of theenvelope protein. Both E1 and E2 appear to have a highly hydrophobicdomain, which apparently anchors the protein within the endoplasmicreticulum and prevents efficient release. Thus, one may wish to deleteportions of the sequence found in one or more of the regions aa170-190,aa260-290 or aa330-380 of E1 (numbering from the beginning of thepolyprotein), and aa660-830 of E2 (see for example FIG. 20-1 of EP388,232). It is likely that at least one of these hydrophobic domainsforms a transmembrane region which is nor essential for antigenicity ofthe protein, and which may thus be deleted without detrimental effect.The best region to delete may be determined by conducting a small numberof deletion experiments within the skill of the ordinary practitioner.Deletion of the hydrophobic 3' end of E2 results in secretion of aportion of the E2 expressed, with sialylation of the secreted protein.

One may use any of a variety of vectors to obtain expression. Lowereukaryotes such as yeast are typically transformed with plasmids usingthe calcium phosphate precipitation method, or are transfected with arecombinant virus. The vectors may replicate within the host cellindependently, or may integrate into the host cell genome. Highereukaryotes may be transformed with plasmids, but are typically infectedwith a recombinant virus, for example a recombinant vaccinia virus.Vaccinia is particularly preferred, as infection with vaccinia haltsexpression of host cell proteins. Presently preferred host cells includeHeLa and plasmacytoma cell lines. In the present system, this means thatE1 and E2 accumulate as the major glycosylated species in the host ER.As the rE1 and rE2 will be the predominant glycoproteins which aremannose-terminated, they may easily be purified from the cells by usinglectins such as Galanthus nivalus agglutinin (GNA) which bind terminalmannose residues.

Proteins which are naturally expressed as mannose-terminatedglycoproteins are relatively rare in mammalian physiology. In mostcases, a mammalian glycoprotein is mannose-terminated only as atransient intermediate in the glycosylation pathway. The fact that HCVenvelope proteins, expressed recombinantly, contain mannose-terminatedglycosylation or (to a lesser degree) N-acetylglucosamine means that HCVproteins and whole virions may be separated and partially purified fromendogenous proteins using lectins specific for terminal mannose orN-acetylglucosamine. The recombinant proteins appear authentic, and arebelieved essentially identical to the envelope proteins found in themature, free virion, or to a form of cell-associated envelope protein.Thus, one may employ lectins such as GNA for mannose-terminatedproteins, and WGA (wheat germ agglutinin) and its equivalents forN-acetylglucosamine-terminated proteins. One may employ lectins bound toa solid phase (e.g., a lectin-Sepharose® column) to separate E1 and E2from cell culture supernatants and other fluids, e.g., for purificationduring the production of antigens for vaccine or immunoassay use.

Alternatively, one may provide a suitable lectin to isolate E1, E2, orHCV virions from fluid or tissue samples from subjects suspected of HCVinfection. As mannose-terminated glycoproteins are relatively rare, sucha procedure should serve to purify the proteins present in a sample,substantially reducing the background. Following binding to lectin, theHCV protein may be detected using anti-HCV antibodies. If whole virionsare present, one may alternatively detect HCV nucleic acids using PCRtechniques or other nucleic acid amplification methods directed towardconserved regions of the HCV genome (for example, the 5' non-codingregion). This method permits isolation and characterization of differingstrains of HCV without regard for antigenic drift or variation, e.g., incases where a new strain is not immunologically crossreactive with thestrain used for preparing antibodies. There are many other ways to takeadvantage of the unique recognition of mannose-terminated glycoproteinsby particular lectins. For example, one may incubate samples suspectedof containing HCV virions or proteins with biotin or avidin-labeledlectins, and precipitate the protein-lectin complex using avidin orbiotin. One may also use lectin affinity for HCV proteins to targetcompounds to virions for therapeutic use, for example by conjugating anantiviral compound to GNA. Alternatively, one may use suitable lectinsto remove mannose-terminated glycoproteins from serum or plasmafractions, thus reducing or eliminating the risk of HCV contamination.

It is presently preferred to isolate E1 and/or E2 asialoglycoproteinsfrom crude cell lysates by incubation with an immobilizedmannose-binding protein, particularly a lectin such as ConA or GNA.Cells are lysed, e.g., by mechanical disruption in a hypoptonic bufferfollowed by centrifugation to prepare a post-nuclear lysate, and furthercentrifuged to obtain a crude microsomal membrane fraction. The crudemembrane fraction is subsequently solubilized in a buffer containing adetergent, such as Triton X-100, NP40, or the like. This detergentextract is further clarified of insoluble particulates bycentrifugation, and the resulting clarified lysate incubated in achromatography column comprising an immobilized mannose-binding protein,preferably GNA bound to a solid support such as agarose or Sepharose®for a period of time sufficient for binding, typically 16 to 20 hours.The suspension is then applied to the column until E1 /E2 begins toappear in the eluent, then incubated in the column for a period of timesufficient for binding, typically about 12-24 hours. The bound materialis then washed with additional buffer containing detergent (e.g., TritonX-100, NP40, or the like), and eluted with mannose to provide purifiedasialoglycoprotein. On elution, it is preferred to elute only untilprotein begins to appear in the eluate, at which point elution is haltedand the column permitted to equilibrate for 2-3 hours before proceedingwith elution of the protein. This is believed to allow sufficient timefor the slow off-rate expected of large protein aggregates. In caseswherein E1 and E2 are expressed together in native form (i.e., withouttruncation of the membrane-binding domain), a substantial fraction ofthe asialoglycoproteins appear as E1:E2 aggregates. When examined byelectron microscopy, a significant portion of these aggregates appear asroughly spherical particles having a diameter of about 40 mn, which isthe size expected for intact virus. These particles appear to beself-assembling subviral particles. These aggregates are expected toexhibit a quaternary structure very similar to the structure ofauthentic HCV virion particles, and thus are expected to serve as highlyimmunogenic vaccines.

The E1/E2 complexes may be further purified by gel chromatography on abasic medium, for example, Fractogel-DEAE or DEAE-Sepharose®. UsingFractogel-DEAE gel chromatography, one may obtain E1 /E2 complexes ofapproximately 60-80% purity. One may further purify E1 by treatment withlysine protease, because E1 has 0-1 Lys residues. Treatment of thecomplex with lysine protease destroys E2, and permits facile separationof E1.

The tissue specificity of HCV, in combination with the observation thatHCV envelope glycoproteins are mannose-terminated, suggests that thevirus employs the mannose receptor or the asialoglycoprotein receptor(ASGR) in order to gain entry into host cells. Mannose receptors arefound on macrophages and hepatic sinusoidal cells, while the ASGR isfound on parenchymal hepatocytes. Thus, it should be possible to cultureHCV by employing host cells which express one or both of thesereceptors. One may either employ primary cell cultures which naturallyexpress the receptor, using conditions under which the receptor ismaintained, or one may transfect another cell line such as HeLa, CHO,COS, and the like, with a vector providing for expression of thereceptor. Cloning of the mannose receptor and its transfection andexpression in fibroblasts has been demonstrated by M. E. Taylor et al, JBiol Chem (1990) 265:12156-62. Cloning and sequencing of the ASGR wasdescribed by K. Drickamer et al, J Biol Chem (1984) 259:770-78 and M.Spiess et al, Proc Nat Acad Sci USA (1985) 82:6465-69; transfection andexpression of functional ASGR in rat HTC cells was described by M.McPhaul and P. Berg, Proc Nat Acad Sci USA (1986) 83:8863-67 and M.McPhaul and P. Berg, Mol Cell Biol (1987) 7:1841-47. Thus, it ispossible to transfect one or both receptors into suitable cell lines,such as CHO, COS, HeLa, and the like, and to use the resulting cells ashosts for propagation of HCV in culture. Serial passaging of HCV in suchcultures should result in development of attenuated strains suitable foruse as live vaccines. It is presently preferred to employ animmortalized cell line transfected with one or both recombinantreceptors.

Immunogenic compositions can be prepared according to methods known inthe art. The present compositions comprise an immunogenic amount of apolypeptide, e.g., E1, E2, or E1/E2 particle compositions, usuallycombined with a pharmaceutically acceptable carrier, preferably furthercomprising an adjuvant. If a "cocktail" is desired, a combination of HCVpolypeptides, such as, for example, E1 plus E2 antigens, can be mixedtogether for heightened efficacy. The virus-like particles of E1/E2aggregates are expected to provide a particularly useful vaccineantigen. Immunogenic compositions may be administered to animals toinduce production of antibodies, either to provide a source ofartibodies or to induce protective immunity in the animal.

Pharmaceutically acceptable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers;and inactive virus particles. Such carriers are well known to those ofordinary skill in the art.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: aluminum hydroxide (alum),N-acetyl-muramyl-L-threonyl-D-isogluta-mine (thr-MDP) as found in U.S.Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE) and RIBI, which contains three components extracted frombacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wallskeleton (MPL+TDM+CWS) in a 2% squalene/Tween® 80 emulsion.Additionally, adjuvants such as Stimulon (Cambridge Bioscience,Worcester, Mass.) may be used. Further, Complete Freund's Adjuvant (CFA)and Incomplete Freund's Adjuvant (IFA) may be used for non-humanapplications and research purposes.

The immunogenic compositions typically will contain pharmaceuticallyacceptable vehicles, such as water, saline, glycerol, ethanol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be included in suchvehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the HCV polypeptide, as well as any other of theabove-mentioned components, as needed. "Immunologically effectiveamount", means that the administration of that amount to an individual,either in a single dose or as part of a series, is effective fortreatment, as defined above. This amount varies depending upon thehealth and physical condition of the individual to be treated, thetaxonomic group of individual to be treated (e.g., nonhuman primate,primate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, the strain of infecting HCV, and other relevant factors. Itis expected that the amount will fall in a relatively broad range thatcan be determined through routine trials.

The self-assembling E1/E2 aggregates may also serve as vaccine carriersto present heterologous (non-HCV) haptens, in the same manner asHepatitis B surface antigen (See European Patent Application 174,444).In this use, the E1/E2 aggregates provide an immunogenic carrier capableof stimulating an immune response to haptens or antigens conjugated tothe aggregate. The antigen may be conjugated either by conventionalchemical methods, or may be cloned into the gene encoding E1 and/or E2at a location corresponding to a hydrophilic region of the protein.

The immunogenic compositions are conventionally administeredparenterally, typically by injection, for example, subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral formulations and suppositories. Dosagetreatment may be a single dose schedule or a multiple dose schedule. Thevaccine may be administered in conjunction with other immunoregulatoryagents.

C. EXAMPLE

The examples presented below are provided as a further guide to thepractitioner of ordinary skill in the art, and are not to be construedas limiting the invention in any way.

Example 1

(Cloning and Expression)

(A) Vectors were constructed from plasmids containing the 5' portion ofthe HCV genome, as described in EP 318,216 and EP 388,232. CassetteHCV(S/B) contains a StuI-BglII DNA fragment encoding the 5' end of thepolyprotein from Met₁ up to Leu₉₀₆, beginning at nucleotide -63 relativeto Met₁. This includes the core protein (C), the E1 protein (alsosometimes referred to as S), the E2 protein (also referred to as NS1),and a 5' portion of the NS2a region. Upon expression of the construct,the individual C, E1 and E2 proteins are produced by proteolyticprocessing.

Cassette HCV(A/B) contains a ApaLI-BglII DNA fragment encoding the 5'end of the polyprotein from Met₁ up to Leu₉₀₆, beginning at nucleotide-6 relative to Met₁. This includes the core protein (C), the E1 protein(also sometimes referred to as S), the E2 protein (also referred to asNS1), and a 5' portion of the NS2a region. Upon expression of theconstruct, the individual C, E1 and E2 proteins are produced byproteolytic processing.

Cassette C-E1(S/B) (a StuI-BamHI portion) contains the 5' end from Met₁up to Ile³⁴⁰ (a BamHI site in the gene). Expression of this cassetteresults in expression of C and a somewhat truncated E1 (E1'). Theportion truncated from the 3' end is a hydrophobic region believed toserve as a translocation signal.

Cassette NS1 (B/B) (a BamHI-BglII portion) contains a small 3' portionof E1 (from Met₃₆₄), all of E2, and a portion of NS2a (to Leu₉₀₆). Inthis construct, the E1 fragment serves as a translocation signal.

Cassette TPA-NS1 employs a human tissue plasminogen activator (tPA)leader as a translocation signal instead of the 3' portion of E1. Thecassette contains a truncated form of E2, from Gly₄₀₆ to Glu₆₆₁, inwhich the hydrophobic 3 end is deleted.

Each cassette was inserted into the vector pGEM3Z (Promega) with andwithout a synthetic β-globin 5' non-coding sequence for transcriptionand translation using T7 and rabbit reticulocyte expression in vitro.Recombinant vaccinia virus (rVV) vectors were prepared by inserting thecassettes into the plasmid pSC11 (obtained from Dr. B. Moss, NIH)followed by recombination with vaccinia virus, as described byCharkrabarty et al, Mol Cell Biol (1985) 5:3403-09.

(B) An alternate expression vector was constructed by inserting HCV(A/B)between the StuI and SpeI sites of pSC59 (obtained from Dr. B. Moss,NIH) followed by recombination with vaccinia virus, as described byCharkrabarty et al, Mol Cell Biol (1985) 5:3403-09.

(C) HeLa S3 cells were collected by centrifugation for 7 minutes at 2000rpm at room temperature in sterile 500 ml centrifuge bottles (JA-10rotor). The pellets were resuspended at a final concentration of 2×10⁷cells/ml in additional culture medium (Joklilk modified MEM Spinnermedium +5% horse serum and Gentamycin) ("spinner medium"). Sonicatedcrude vv/SC59-HCV virus stock was added at a multiplicity of infectionof 8 pfu/cell, and the mixture stirred at 37° C. for 30 minutes. Theinfected cells were then transferred to a spinner flask containing 8liters spinner medium and incubated for 3 days at 37° C.

The cultured cells were then collected by centrifugation, and thepellets resuspended in buffer (10 mM Tris-HCl, pH 9.0, 15 ml). The cellswere then homogenized using a 40 ml Dounce Homogenizer (50 strokes), andthe nuclei pelleted by centrifugation (5 minutes, 1600 rpm, 4° C., JA-20rotor). The nuclear pellets were resuspended in Tris buffer (4 ml),rehomogenized, and pelleted again, pooling all supernatants.

The pooled lysate was divided into 10 ml aliquots and sonicated 3×30minutes in a cuphorn sonicator at medium power. The sonicated lysate (15ml) was layered onto 17 ml sucrose cushions (36%) in SW28 centrifugetubes, and centrifuged at 13,500 rpm for 80 minutes at 4° C. to pelletthe virus. The virus pellet was resuspended in 1 ml of Tris buffer (1 mMTris HCl, pH 9.0) and frozen at -80° C.

Example 2

(Comparison of In Vitro and In Vivo Products)

(A) E1 and E2 were expressed both in vitro and in vivo and ³⁵ S-Metlabeled using the vectors described in Example 1 above. BSC-40 and HeLacells were infected with the rVV vectors for in vivo expression. Boththe medium and the cell lysates were examined for recombinant proteins.The products were immunoprecipitated using human HCV immune serum, whilein vitro proteins were analyzed directly. The resulting proteins wereanalyzed by SDS-PAGE.

The reticulocyte expression system (pGEM3Z with HCV(S/B) or HCV(A/B))produced C, E1 and E2 proteins having molecular weights of approximately18 kD, 35 kD, and 72 kD, respectively. Lysates from BSC-40 and HeLacells transfected with rVV containing HCV(S/B), HCV(A/B) or C-E1 (S/B)exhibited the same proteins. Because the reticulocyte system does notprovide efficient golgi processing and therefore does not provide sialicacid, the fact that both in vitro and in vivo products exhibitedidentical mobilities suggests that the proteins are not sialylated invivo. Only the rVV vector containing TPA-NS1 resulted in anyextracellular secretion of E2, which exhibited an altered mobilityconsistent with sialylation.

(B) HCV(S/B) was expressed in vitro and incubated with a panel ofbiotinylated lectins: GNA, SNA, PNA, WGA, and ConA. Followingincubation, the complexes were collected on avidin-acrylic beads,washed, eluted with Laemmli sample buffer, and analyzed by SDS-PAGE. Theresults showed that E1 and E2 bound to GNA and ConA, which indicates thepresence of mannose. GNA binds to terminal mannose groups, while ConAbinds to any α-linked mannose. The lack of binding to SNA, PNA, and WGAindicates that none of the proteins contained sialic acid,galactose-N-acetylgalactosamine, or N-acetylglucosamine.

(C) Radiolabeled E1 and E2 were produced in BSC-40 cells by infectionwith rVV containing HCV(S/B) (vv/SClI-HCV), and immunoprecipitated withhuman HCV⁺ immune serum. One half of the immunoprecipitated material wastreated overnight with neuraminidase to remove any sialic acid.Following treatment, the treated and untreated proteins were analyzed bySDS-PAGE. No significant difference in mobility was observed, indicatinglack of sialylation in vivo.

(D) Radiolabeled E1 and E2 were produced in BSC-40 cells by infectionwith rVV containing HCV(A/B) (vv/SC59-HCV), and eitherimmunoprecipitated with human HCV⁺ serum, or precipitated usingbiotinylated GNA lectin linked to acrylic beads, using vv/SC11 free ofHCV sequences as control. The precipitates were analyzed by SDS-PAGE.The data demonstrated that E1 and E2 were the major species ofmannose-terminated proteins in vv/SC59-HCV infected cells. GNA was asefficient as human antisera in precipitating E1 and E2 from cell culturemedium. A 25 kD component was observed, but appears to be specific tovaccinia-infected cells.

Example 3

(Purification Using Lectin)

(A) HeLa S3 cells were inoculated with purified high-titer w/SC59-HCVvirus stock at a multiplicity of infection of 5 pfu/cell, and themixture stirred at 37° C. for 30 minutes. The infected cells were thentransferred to a spinner flask containing 8 liters spinner medium andincubated for 3 days at 37° C. The cells were collected again bycentrifugation and resuspended in hypotonic buffer (20 mM HEPES, 10 mMNaCl, 1 mM MgCl₂, 120 ml) on ice. The cells were then homogenized byDounce Homogenizer (50 strokes), and the nuclei pelleted bycentrifugation (5 minutes, 1600 rpm, 4° C., JA-20 rotor). The pelletswere pooled, resuspended in 48 ml hypotonic buffer, rehomogenized,recentrifuged, pooled again, and frozen at -80° C.

The frozen supernatants were then thawed, and the microsomal membranefraction of the post-nuclear lysate isolated by centrifuging for 20minutes in a JA-20 rotor at 13,500 rpm at 4° C. The supernatant wasremoved by aspiration.

The pellets were taken up in 96 ml detergent buffer (20 mM Tris-HCl, 100mM NaCl, 1 mM EDTA, 1 mM DDT, 0.5% Triton X-100, pH 7.5) and homogenized(50 strokes). The product was clarified by centrifugation for 20 minutesat 13,500 rpm, 4° C., and the supernatants collected.

A GNA-agarose column (1 cm×3 cm, 3 mg GNA/ml beads, 6 ml bed volume,Vector Labs, Burlingame, Calif.) was pre-equilibrated with detergentbuffer. The supernatant sample was applied to the column withrecirculation at a flow rate of 1 ml/min for 16-20 hours at 4° C. Thecolumn was then washed with detergent buffer.

The purified E1/E2 proteins were eluted with α-D-mannoside (0.9 M indetergent buffer) at a flow rate of 0.5 ml/minute. E1 ution was haltedat the appearance of E1/E2 in the eluent, and the column allowed toreequilibrate for 2-3 hours. Fractions were analyzed by Western blot andsilver staining. Peak fractions were pooled and UV-irradiated toinactivate any residual vaccinia virus.

B GNA-agarose purified E1 and E2 asialoglycoproteins were sedimentedthrough 20-60% glycerol gradients. The gradients were fractionated andproteins were analyzed by SDS-PAGE and western blotting. Blots wereprobed with GNA for identification of E1 and E2. The results indicatethe presence of a E1:E2 heterodimer which sediments at the expected rate(i.e., a position characteristic of a 110 kD protein). Larger aggregatesof HCV envelope proteins also are apparent. E2:E2 homodimers also wereapparent. E2 appeared to be over-represented in the larger speciesrelative to E1, although discrete E1:E2 species also were detected. Thelarger aggregates sedimented significantly faster than the thyroglobulinmarker.

(C) GNA-agarose purified E1 and E2 were sedimented through 20-60%glycerol gradients containing 1 mM EDTA. Fractions were analyzed bySDS-PAGE with and without β-mercaptoethanol (βME). Little or nodifference in the apparent abundance of E1 and E2 in the presence orabsence of ME was observed, indicating the absence of disulfide linksbetween heterodimers.

(D) E1/E2 complexes (approximately 40% pure) were analyzed on a CoulterDM-4 sub-micron particle analyzer. Material in the 20-60 nm range wasdetected.

(E) E1/E2 complexes (approximately 40% pure) were analyzed by electronmicroscopy using negative staining with phosphotungstic acid. Theelectron micrograph revealed the presence of particles having aspherical appearance and a diameter of about 40 nmn. E1/E2 complexeswere incubated with HCV⁺ human immune serum, then analyzed by EM withnegative staining. Antibody complexes containing large aggregates andsmaller particles were observed.

Example 4

(Chromatographic Purification)

(A) The GNA lectin-purified material prepared as described in Example 3(0.5-0.8 ml) was diluted 10× with buffer A (20 mM Tris-Cl buffer, pH8.0, 1 mM EDTA), and applied to a 1.8×1.5 cm column of Fractogel EMDDEAE-650 (EM Separations, Gibbstown, N.J., cat. no. 16883) equilibratedin buffer A. The protein fraction containing E1/E2 was eluted with thesame buffer at a flow rate of 0.2 ml/minute, and 1 ml fractionscollected. Fractions containing E1 and E2 (determined by SDS-PAGE) werepooled and stored at -80° C.

(B) The material purified in part (A) above has a purity of 60-80%, asestimated by SDS-PAGE. The identification of the putative E1 and E2bands was confirmed by N-terminal sequence analysis after using atransfer technique. For the purpose, the fractogel-DEAE purified E1/E2material was reduced by addition of Laemmli buffer (pH 6.8, 0.06 MTris-Cl, 2.3% SDS, 10% glycerol, 0.72 M β-mercaptoethanol) and boiledfor 3 minutes. The sample was then loaded onto a 10% polyacrylamide gel.After SDS-PAGE, the protein was transferred to a polyvinylidenedifluoride (PVDF) 0.2 μm membrane (Bio-Rad Laboratories, Richmond,Calif.). The respective putative E1 and E2 protein bands were excisedfrom the blot and subjected to N-terminal amino acid analysis, althoughno special care was taken to prevent amino-terminal blockage duringpreparation of the material. The first 15 cycles revealed that the E1sample had a sequenceTyr-Gln-Val-Arg-X-Ser-Thr-Gly-X-Tyr-His-Val-X-Asn-Asp (SEQ. ID NO:1),while the sequence of E2 wasTHr-His-Val-Thr-Gly-X-X-Ala-Gly-His-X-Val-X-Gly-Phe (SEQ. ID NO:2). Thisamino acid sequence data is in agreement with that expected from thecorresponding DNA sequences.

The E1/E2 product purified above by fractogel-DEAE chromatography isbelieved to be aggregated as evidenced by the fact that a large amountof E1 and E2 coelutes in the void volume region of a gel permeationchromatographic Bio-Sil TSK-4000 SW column. This indicates that undernative conditions a significant amount of the E1/E2 complex has amolecular weight of at least 800 kD. E1/E2 material having a molecularweight of about 650 kD was also observed.

Example 5

(Additional Cloning and Expression)

(A) The following cassettes containing 5' portions of the HCVpolyprotein were inserted into the vector pGEM4Z (Promega) with andwithout a synthetic yellow fever virus 5' non-coding sequence and alsointo recombinant vaccinia virus (rVV) vectors (as described in Example1A). Cassette C5p-1 contains a fragment encoding the 5' end of thepolyprotein from Met₁ to Trp₁₀₇₉, beginning at nucleotide -275 relativeto Met₁, with EcoRI linkers on the 5' and 3' ends. Cassette C5p-3contains an fragment encoding the 5' end of the polyprotein from Met₁ toTrp₁₀₇₉, with EcoRI linkers on the 5' and 3' ends. Both cassettes encodeC, E1 and E2 proteins and a 5' portion of the NS2 protein.

(B) The following cassettes containing 5' portions of the HCVpolyprotein were inserted into the vector pSC59 followed byrecombination with vaccinia virus (described in Example 1B). CassetteHCV(Poly) contains a blunt-ended StuI-BglII fragment encoding the 5' endof the polyprotein from Met₁ to Asp₉₆₆ beginning at nucleotide -65relative to Met₁. This construct expresses C, E1 and E2 proteins, and a5' portion of the NS2 protein.

Cassette HCV(5C/SB) contains a blunt-ended StuI-BamHI fragment encodingthe 5' end of the polyprotein from Met₁ to Ile₃₄₀ beginning atnucleotide -65 relative to Met₁. This construct expresses the C proteinand a truncated E1 protein.

Cassette HCV(6C/SS) contains a SalU(blunted)-EcoRI fragment encoding the5' end of the polyprotein from Met₁ to Asp₃₈₂ wherein Ser₂ is replacedwith Gly₂. This construct expresses the C protein and a truncated E1protein.

Cassette HCV(E12C/B) contains a blunt-ended ClaI-BglII fragment encodinga portion of the polyprotein from Met₁₃₄ to ASP₉₆₆ inserted into anEcoRI blunted SC59 vector.

Cassette HCV(E1/S) contains a blunt-ended ClaI/SalI fragment encoding aportion of the polyprotein from Met₁₃₄ to Val₃₈₁, inserted into an EcoRIblunted SC59 vector.

(C) HeLa S3 cells were collected by centrifugation for 7 minutes at 2000rpm at 4° C. in sterile 250 ml centrifuge bottles. The pellets wereresuspended at a final concentration of 5×10⁶ cells/ml in Gey's balancedSalt Solution (GBSS). Sonicated crude vv/SC59-HCV virus stock was addedat a multiplicity of infection of 0.5 pfu/cell, and the mixture stirredat 37° C. for 1-2 hours. The infected cells were then transferred at afinal concentration of 10⁶ cells/ml to a spinner flask containing 1liter culture medium (Joklik MEM+10% fetal bovine serum+non-essentialamino acids, vitamins, pen/strep) and incubated for 3 days at 37° C.

The cultured cells were then collected by centrifugation, and thepellets resuspended in buffer (10 mM Tris-HCl, pH 9.0, 15 ml). The cellswere then homogenized using a 40 ml Dounce Homogenizer (50 strokes), andthe nuclei pelleted by centrifugation (5 minutes, 1600 rpm, 4° C., JA-20rotor). The nuclear pellets were resuspended in Tris buffer (4 ml),rehomogenized, and pelleted again, pooling all supernatants.

The pooled lysate was divided into 5 ml aliquots, and 0.1 volume of 2.5mg/ml trypsin added and incubated at 37° C. for 30 minutes. The aliquotswere then sonicated 3×30 seconds in a cuphorn sonicator at medium power.The sonicated lysate was used as the crude stock.

Example 6

(Additional Comparison of In Vitro and In Vivo Products)

(A) E1 and E2 were expressed both in vitro and in vivo and ³⁵ S-Metlabeled using the vectors described in Example 5 above and theprocedures described in Example 2 above. BSC-40 and HeLa cells wereinfected with the rVV vectors for in vivo expression. Both the mediumand the cell lysates were examined for recombinant proteins. Theproducts were immunoprecipitated using human HCV immune serum or rabbitor goat anti-HCV antiserum, while in vitro proteins were analyzeddirectly. The resulting proteins were analyzed by SDS-PAGE and EndoHdigestion.

Example 7

(Additional Lectin Purification)

(A) HeLa S3 cells were inoculated with purified high-titer vv/SC59-HCVvirus stock (HCV(Poly) or HCV(E12C/B) as described in Example 5 above)at a multiplicity of infection of 1 pfu/cell, and the mixture stirred at37° C. for 1-2 hours. The infected cells were then transferred tospinner flasks containing 1 liter culture medium (see Example 5, supra)and incubated for 2 days at 37° C. A total of 10 liters of cells werecollected by centrifugation and resuspended in hypotonic buffer (20 mMHEPES, 10 mM NaCl, 1 mM MgCl₂, 120 ml) containing protease inhibitors(PMSF and pepstatin A) on ice. The cells were then homogenized in a 40ml homogenizer in two batches, pelleted by centrifugation (20 minutes,12,000 rpm), and re-suspended and re-homogenized. Each pellet wasresuspended in approximately 10 ml 25 mM NaPO₄ (pH 6.8) in ahomogenizer, and an equal volume of 4% Triton X-100 in 100 mM NaPO₄ (pH6.8) added. The pelleted cells were homogenized with 20 strokes, spun at12,000 rpm for 15 minutes (4 tubes/pellet), and the supernatant saved.The resuspension, Triton addition and centrifugation steps wererepeated, and the saved supernatants combined, and frozen at -80° C.

The frozen supernatants were thawed, spun at 12,000 rpm for 15 minutes,combined and held at 4° C. A GNA-agarose column (1 cm×3 cm, 3 mg GNA/mlbeads, 6 ml bed volume, Vector Labs, Burlingame, Calif.) waspre-equilibrated with detergent buffer (2% Triton X-100 in 50 mM NaPO₄,pH 6.8). The supernatant sample was applied to the column withrecirculation at a flow rate of 1 ml/min for 16-20 hours at 4° C. Thecolumn was then washed with detergent buffer, followed by 30 ml each of:Buffer A (1M NaCl, 20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100); Buffer B(20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100); Buffer D (0.2Mmethyl-α-D-mannopyranoside (mmp), 20 mM NaPO₄ (pH 6.0), 0.1% TritonX-100); Buffer E (1M mmp, 20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100);Buffer F (1M mmp, 1M NaCl, 20 mM NaPO₄ (pH 6.0), 0.1% Triton X-100).Purified E1/E2 proteins come off as eluted material in Buffers D and E,which were collected separately and analyzed by SDS-PAGE.

Example 8

(Additional Chromatographic Purification)

(A) The GNA lectin-purified material prepared as described in Example 7was applied to a column of S-Sepharose Fast Flow (Pharrnacia)equilibrated in buffer B (see Example 7). The column was washed withBuffer B, then eluted with Buffer 1 (0.5M NaCl, 20 mM NaPO₄ (pH 6.0),0.1% Triton X-100) and Buffer 2 (1M NaCl, 20 mM NaPO₄ (pH 6.0), 0.1%Triton X-100). Fractions containing E1 and E2 (determined by SDS-PAGE)were pooled and stored at -80° C.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Tyr Gln Val Arg Xaa Ser Thr Gly Xaa Tyr Hi - #s Val Xaa Asn Asp          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Thr His Val Thr Gly Xaa Xaa Ala Gly His Xa - #a Val Xaa Gly Phe          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2955 amino - #acids                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Arg or Lys"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Asn or Thr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 176                                                             (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Ile or Thr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 334                                                             (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Met or Val"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 603                                                             (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Ile or Leu"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 848                                                             (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Asn or Tyr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1114                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Pro or Ser"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1117                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Ser or Thr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1276                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Leu or Pro"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1454                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Cys or Tyr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1471                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Ser or Thr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1877                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Glu or Gly"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1948                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = His or Leu"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1949                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Cys or Ser"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2021                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Gly or Val"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2349                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Ser or Thr"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2385                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa =Phe or Tyr"                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2386                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Ala or Ser"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2502                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Phe or Leu"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2690                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Gly or Arg"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2921                                                            (D) OTHER INFORMATION: - #/note= "There is a heterogeneity at                      this loca - #tion; Xaa = Arg or Gly"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Ser Thr Asn Pro Lys Pro Gln Xaa Lys Xa - #a Lys Arg Asn Thr Asn      1               5   - #                10  - #                15               - - Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gl - #y Gly Gln Ile Val Gly                  20      - #            25      - #            30                   - - Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Ar - #g Leu Gly Val Arg Ala              35          - #        40          - #        45                       - - Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Ar - #g Gly Arg Arg Gln Pro          50              - #    55              - #    60                           - - Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Th - #r Trp Ala Gln Pro Gly      65                  - #70                  - #75                  - #80        - - Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cy - #s Gly Trp Ala Gly Trp                      85  - #                90  - #                95               - - Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Tr - #p Gly Pro Thr Asp Pro                  100      - #           105      - #           110                  - - Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Il - #e Asp Thr Leu Thr Cys              115          - #       120          - #       125                      - - Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Le - #u Val Gly Ala Pro Leu          130              - #   135              - #   140                          - - Gly Ser Ala Ala Arg Ala Leu Ala His Gly Va - #l Arg Val Leu Glu Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gl - #y Cys Ser Phe Ser        Xaa                                                                                             165  - #               170  - #               175             - - Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Va - #l Pro Ala Ser Ala Tyr                  180      - #           185      - #           190                  - - Gln Val Arg Asn Ser Thr Gly Leu Tyr His Va - #l Thr Asn Asp Cys Pro              195          - #       200          - #       205                      - - Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Al - #a Ile Leu His Thr Pro          210              - #   215              - #   220                          - - Gly Cys Val Pro Cys Val Arg Glu Gly Asn Al - #a Ser Arg Cys Trp Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gl - #y Lys Leu Pro Ala        Thr                                                                                             245  - #               250  - #               255             - - Gln Leu Arg Arg His Ile Asp Leu Leu Val Gl - #y Ser Ala Thr Leu Cys                  260      - #           265      - #           270                  - - Ser Ala Leu Tyr Val Gly Asp Leu Cys Gly Se - #r Val Phe Leu Val Gly              275          - #       280          - #       285                      - - Gln Leu Phe Thr Phe Ser Pro Arg Arg His Tr - #p Thr Thr Gln Gly Cys          290              - #   295              - #   300                          - - Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gl - #y His Arg Met Ala Trp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Met Met Met Asn Trp Ser Pro Thr Thr Al - #a Leu Val Xaa Ala        Gln                                                                                             325  - #               330  - #               335             - - Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Me - #t Ile Ala Gly Ala His                  340      - #           345      - #           350                  - - Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Se - #r Met Val Gly Asn Trp              355          - #       360          - #       365                      - - Ala Lys Val Leu Val Val Leu Leu Leu Phe Al - #a Gly Val Asp Ala Glu          370              - #   375              - #   380                          - - Thr His Val Thr Gly Gly Ser Ala Gly His Th - #r Val Ser Gly Phe Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ser Leu Leu Ala Pro Gly Ala Lys Gln Asn Va - #l Gln Leu Ile Asn        Thr                                                                                             405  - #               410  - #               415             - - Asn Gly Ser Trp His Leu Asn Ser Thr Ala Le - #u Asn Cys Asn Asp Ser                  420      - #           425      - #           430                  - - Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Ty - #r His His Lys Phe Asn              435          - #       440          - #       445                      - - Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cy - #s Arg Pro Leu Thr Asp          450              - #   455              - #   460                          - - Phe Asp Gln Gly Trp Gly Pro Ile Ser Tyr Al - #a Asn Gly Ser Gly Pro      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asp Gln Arg Pro Tyr Cys Trp His Tyr Pro Pr - #o Lys Pro Cys Gly        Ile                                                                                             485  - #               490  - #               495             - - Val Pro Ala Lys Ser Val Cys Gly Pro Val Ty - #r Cys Phe Thr Pro Ser                  500      - #           505      - #           510                  - - Pro Val Val Val Gly Thr Thr Asp Arg Ser Gl - #y Ala Pro Thr Tyr Ser              515          - #       520          - #       525                      - - Trp Gly Glu Asn Asp Thr Asp Val Phe Val Le - #u Asn Asn Thr Arg Pro          530              - #   535              - #   540                          - - Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Me - #t Asn Ser Thr Gly Phe      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Thr Lys Val Cys Gly Ala Pro Pro Cys Val Il - #e Gly Gly Ala Gly        Asn                                                                                             565  - #               570  - #               575             - - Asn Thr Leu His Cys Pro Thr Asp Cys Phe Ar - #g Lys His Pro Asp Ala                  580      - #           585      - #           590                  - - Thr Tyr Ser Arg Cys Gly Ser Gly Pro Trp Xa - #a Thr Pro Arg Cys Leu              595          - #       600          - #       605                      - - Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pr - #o Cys Thr Ile Asn Tyr          610              - #   615              - #   620                          - - Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gl - #y Val Glu His Arg Leu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Ar - #g Cys Asp Leu Glu        Asp                                                                                             645  - #               650  - #               655             - - Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Le - #u Thr Thr Thr Gln Trp                  660      - #           665      - #           670                  - - Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pr - #o Ala Leu Ser Thr Gly              675          - #       680          - #       685                      - - Leu Ile His Leu His Gln Asn Ile Val Asp Va - #l Gln Tyr Leu Tyr Gly          690              - #   695              - #   700                          - - Val Gly Ser Ser Ile Ala Ser Trp Ala Ile Ly - #s Trp Glu Tyr Val Val      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Va - #l Cys Ser Cys Leu        Trp                                                                                             725  - #               730  - #               735             - - Met Met Leu Leu Ile Ser Gln Ala Glu Ala Al - #a Leu Glu Asn Leu Val                  740      - #           745      - #           750                  - - Ile Leu Asn Ala Ala Ser Leu Ala Gly Thr Hi - #s Gly Leu Val Ser Phe              755          - #       760          - #       765                      - - Leu Val Phe Phe Cys Phe Ala Trp Tyr Leu Ly - #s Gly Lys Trp Val Pro          770              - #   775              - #   780                          - - Gly Ala Val Tyr Thr Phe Tyr Gly Met Trp Pr - #o Leu Leu Leu Leu Leu      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Leu Ala Leu Pro Gln Arg Ala Tyr Ala Leu As - #p Thr Glu Val Ala        Ala                                                                                             805  - #               810  - #               815             - - Ser Cys Gly Gly Val Val Leu Val Gly Leu Me - #t Ala Leu Thr Leu Ser                  820      - #           825      - #           830                  - - Pro Tyr Tyr Lys Arg Tyr Ile Ser Trp Cys Le - #u Trp Trp Leu Gln Xaa              835          - #       840          - #       845                      - - Phe Leu Thr Arg Val Glu Ala Gln Leu His Va - #l Trp Ile Pro Pro Leu          850              - #   855              - #   860                          - - Asn Val Arg Gly Gly Arg Asp Ala Val Ile Le - #u Leu Met Cys Ala Val      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - His Pro Thr Leu Val Phe Asp Ile Thr Lys Le - #u Leu Leu Ala Val        Phe                                                                                             885  - #               890  - #               895             - - Gly Pro Leu Trp Ile Leu Gln Ala Ser Leu Le - #u Lys Val Pro Tyr Phe                  900      - #           905      - #           910                  - - Val Arg Val Gln Gly Leu Leu Arg Phe Cys Al - #a Leu Ala Arg Lys Met              915          - #       920          - #       925                      - - Ile Gly Gly His Tyr Val Gln Met Val Ile Il - #e Lys Leu Gly Ala Leu          930              - #   935              - #   940                          - - Thr Gly Thr Tyr Val Tyr Asn His Leu Thr Pr - #o Leu Arg Asp Trp Ala      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - His Asn Gly Leu Arg Asp Leu Ala Val Ala Va - #l Glu Pro Val Val        Phe                                                                                             965  - #               970  - #               975             - - Ser Gln Met Glu Thr Lys Leu Ile Thr Trp Gl - #y Ala Asp Thr Ala Ala                  980      - #           985      - #           990                  - - Cys Gly Asp Ile Ile Asn Gly Leu Pro Val Se - #r Ala Arg Arg Gly Arg              995          - #       1000          - #      1005                     - - Glu Ile Leu Leu Gly Pro Ala Asp Gly Met Va - #l Ser Lys Gly Trp Arg          1010             - #   1015              - #  1020                         - - Leu Leu Ala Pro Ile Thr Ala Tyr Ala Gln Gl - #n Thr Arg Gly Leu Leu      1025                1030 - #                1035 - #               1040        - - Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg As - #p Lys Asn Gln Val Glu                      1045 - #               1050  - #              1055             - - Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gl - #n Thr Phe Leu Ala Thr                  1060     - #           1065      - #          1070                 - - Cys Ile Asn Gly Val Cys Trp Thr Val Tyr Hi - #s Gly Ala Gly Thr Arg              1075         - #       1080          - #      1085                     - - Thr Ile Ala Ser Pro Lys Gly Pro Val Ile Gl - #n Met Tyr Thr Asn Val          1090             - #   1095              - #  1100                         - - Asp Gln Asp Leu Val Gly Trp Pro Ala Xaa Gl - #n Gly Xaa Arg Ser Leu      1105                1110 - #                1115 - #               1120        - - Thr Pro Cys Thr Cys Gly Ser Ser Asp Leu Ty - #r Leu Val Thr Arg His                      1125 - #               1130  - #              1135             - - Ala Asp Val Ile Pro Val Arg Arg Arg Gly As - #p Ser Arg Gly Ser Leu                  1140     - #           1145      - #          1150                 - - Leu Ser Pro Arg Pro Ile Ser Tyr Leu Lys Gl - #y Ser Ser Gly Gly Pro              1155         - #       1160          - #      1165                     - - Leu Leu Cys Pro Ala Gly His Ala Val Gly Il - #e Phe Arg Ala Ala Val          1170             - #   1175              - #  1180                         - - Cys Thr Arg Gly Val Ala Lys Ala Val Asp Ph - #e Ile Pro Val Glu Asn      1185                1190 - #                1195 - #               1200        - - Leu Glu Thr Thr Met Arg Ser Pro Val Phe Th - #r Asp Asn Ser Ser Pro                      1205 - #               1210  - #              1215             - - Pro Val Val Pro Gln Ser Phe Gln Val Ala Hi - #s Leu His Ala Pro Thr                  1220     - #           1225      - #          1230                 - - Gly Ser Gly Lys Ser Thr Lys Val Pro Ala Al - #a Tyr Ala Ala Gln Gly              1235         - #       1240          - #      1245                     - - Tyr Lys Val Leu Val Leu Asn Pro Ser Val Al - #a Ala Thr Leu Gly Phe          1250             - #   1255              - #  1260                         - - Gly Ala Tyr Met Ser Lys Ala His Gly Ile As - #p Xaa Asn Ile Arg Thr      1265                1270 - #                1275 - #               1280        - - Gly Val Arg Thr Ile Thr Thr Gly Ser Pro Il - #e Thr Tyr Ser Thr Tyr                      1285 - #               1290  - #              1295             - - Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gl - #y Gly Ala Tyr Asp Ile                  1300     - #           1305      - #          1310                 - - Ile Ile Cys Asp Glu Cys His Ser Thr Asp Al - #a Thr Ser Ile Leu Gly              1315         - #       1320          - #      1325                     - - Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Al - #a Gly Ala Arg Leu Val          1330             - #   1335              - #  1340                         - - Val Leu Ala Thr Ala Thr Pro Pro Gly Ser Va - #l Thr Val Pro His Pro      1345                1350 - #                1355 - #               1360        - - Asn Ile Glu Glu Val Ala Leu Ser Thr Thr Gl - #y Glu Ile Pro Phe Tyr                      1365 - #               1370  - #              1375             - - Gly Lys Ala Ile Pro Leu Glu Val Ile Lys Gl - #y Gly Arg His Leu Ile                  1380     - #           1385      - #          1390                 - - Phe Cys His Ser Lys Lys Lys Cys Asp Glu Le - #u Ala Ala Lys Leu Val              1395         - #       1400          - #      1405                     - - Ala Leu Gly Ile Asn Ala Val Ala Tyr Tyr Ar - #g Gly Leu Asp Val Ser          1410             - #   1415              - #  1420                         - - Val Ile Pro Thr Ser Gly Asp Val Val Val Va - #l Ala Thr Asp Ala Leu      1425                1430 - #                1435 - #               1440        - - Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Va - #l Ile Asp Xaa Asn Thr                      1445 - #               1450  - #              1455             - - Cys Val Thr Gln Thr Val Asp Phe Ser Leu As - #p Pro Thr Phe Xaa Ile                  1460     - #           1465      - #          1470                 - - Glu Thr Ile Thr Leu Pro Gln Asp Ala Val Se - #r Arg Thr Gln Arg Arg              1475         - #       1480          - #      1485                     - - Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile As - #n Arg Phe Val Ala Pro          1490             - #   1495              - #  1500                         - - Gly Glu Arg Pro Ser Gly Met Phe Asp Ser Se - #r Val Leu Cys Glu Cys      1505                1510 - #                1515 - #               1520        - - Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu Th - #r Pro Ala Glu Thr Thr                      1525 - #               1530  - #              1535             - - Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gl - #y Leu Pro Val Cys Gln                  1540     - #           1545      - #          1550                 - - Asp His Leu Glu Phe Trp Glu Gly Val Phe Th - #r Gly Leu Thr His Ile              1555         - #       1560          - #      1565                     - - Asp Ala His Phe Leu Ser Gln Thr Lys Gln Se - #r Gly Glu Asn Leu Pro          1570             - #   1575              - #  1580                         - - Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys Al - #a Arg Ala Gln Ala Pro      1585                1590 - #                1595 - #               1600        - - Pro Pro Ser Trp Asp Gln Met Trp Lys Cys Le - #u Ile Arg Leu Lys Pro                      1605 - #               1610  - #              1615             - - Thr Leu His Gly Pro Thr Pro Leu Leu Tyr Ar - #g Leu Gly Ala Val Gln                  1620     - #           1625      - #          1630                 - - Asn Glu Ile Thr Leu Thr His Pro Val Thr Ly - #s Tyr Ile Met Thr Cys              1635         - #       1640          - #      1645                     - - Met Ser Ala Asp Leu Glu Val Val Thr Ser Th - #r Trp Val Leu Val Gly          1650             - #   1655              - #  1660                         - - Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Le - #u Ser Thr Gly Cys Val      1665                1670 - #                1675 - #               1680        - - Val Ile Val Gly Arg Val Val Leu Ser Gly Ly - #s Pro Ala Ile Ile Pro                      1685 - #               1690  - #              1695             - - Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Gl - #u Met Glu Glu Cys Ser                  1700     - #           1705      - #          1710                 - - Gln His Leu Pro Tyr Ile Glu Gln Gly Met Me - #t Leu Ala Glu Gln Phe              1715         - #       1720          - #      1725                     - - Lys Gln Lys Ala Leu Gly Leu Leu Gln Thr Al - #a Ser Arg Gln Ala Glu          1730             - #   1735              - #  1740                         - - Val Ile Ala Pro Ala Val Gln Thr Asn Trp Gl - #n Lys Leu Glu Thr Phe      1745                1750 - #                1755 - #               1760        - - Trp Ala Lys His Met Trp Asn Phe Ile Ser Gl - #y Ile Gln Tyr Leu Ala                      1765 - #               1770  - #              1775             - - Gly Leu Ser Thr Leu Pro Gly Asn Pro Ala Il - #e Ala Ser Leu Met Ala                  1780     - #           1785      - #          1790                 - - Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Th - #r Ser Gln Thr Leu Leu              1795         - #       1800          - #      1805                     - - Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gl - #n Leu Ala Ala Pro Gly          1810             - #   1815              - #  1820                         - - Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Al - #a Gly Ala Ala Ile Gly      1825                1830 - #                1835 - #               1840        - - Ser Val Gly Leu Gly Lys Val Leu Ile Asp Il - #e Leu Ala Gly Tyr Gly                      1845 - #               1850  - #              1855             - - Ala Gly Val Ala Gly Ala Leu Val Ala Phe Ly - #s Ile Met Ser Gly Glu                  1860     - #           1865      - #          1870                 - - Val Pro Ser Thr Xaa Asp Leu Val Asn Leu Le - #u Pro Ala Ile Leu Ser              1875         - #       1880          - #      1885                     - - Pro Gly Ala Leu Val Val Gly Val Val Cys Al - #a Ala Ile Leu Arg Arg          1890             - #   1895              - #  1900                         - - His Val Gly Pro Gly Glu Gly Ala Val Gln Tr - #p Met Asn Arg Leu Ile      1905                1910 - #                1915 - #               1920        - - Ala Phe Ala Ser Arg Gly Asn His Val Ser Pr - #o Thr His Tyr Val Pro                      1925 - #               1930  - #              1935             - - Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Il - #e Xaa Xaa Ser Leu Thr                  1940     - #           1945      - #          1950                 - - Val Thr Gln Leu Leu Arg Arg Leu His Gln Tr - #p Ile Ser Ser Glu Cys              1955         - #       1960          - #      1965                     - - Thr Thr Pro Cys Ser Gly Ser Trp Leu Arg As - #p Ile Trp Asp Trp Ile          1970             - #   1975              - #  1980                         - - Cys Glu Val Leu Ser Asp Phe Lys Thr Trp Le - #u Lys Ala Lys Leu Met      1985                1990 - #                1995 - #               2000        - - Pro Gln Leu Pro Gly Ile Pro Phe Val Ser Cy - #s Gln Arg Gly Tyr Lys                      2005 - #               2010  - #              2015             - - Gly Val Trp Arg Xaa Asp Gly Ile Met His Th - #r Arg Cys His Cys Gly                  2020     - #           2025      - #          2030                 - - Ala Glu Ile Thr Gly His Val Lys Asn Gly Th - #r Met Arg Ile Val Gly              2035         - #       2040          - #      2045                     - - Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Th - #r Phe Pro Ile Asn Ala          2050             - #   2055              - #  2060                         - - Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Al - #a Pro Asn Tyr Thr Phe      2065                2070 - #                2075 - #               2080        - - Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr Va - #l Glu Ile Arg Gln Val                      2085 - #               2090  - #              2095             - - Gly Asp Phe His Tyr Val Thr Gly Met Thr Th - #r Asp Asn Leu Lys Cys                  2100     - #           2105      - #          2110                 - - Pro Cys Gln Val Pro Ser Pro Glu Phe Phe Th - #r Glu Leu Asp Gly Val              2115         - #       2120          - #      2125                     - - Arg Leu His Arg Phe Ala Pro Pro Cys Lys Pr - #o Leu Leu Arg Glu Glu          2130             - #   2135              - #  2140                         - - Val Ser Phe Arg Val Gly Leu His Glu Tyr Pr - #o Val Gly Ser Gln Leu      2145                2150 - #                2155 - #               2160        - - Pro Cys Glu Pro Glu Pro Asp Val Ala Val Le - #u Thr Ser Met Leu Thr                      2165 - #               2170  - #              2175             - - Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gl - #y Arg Arg Leu Ala Arg                  2180     - #           2185      - #          2190                 - - Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Al - #a Ser Gln Leu Ser Ala              2195         - #       2200          - #      2205                     - - Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn Hi - #s Asp Ser Pro Asp Ala          2210             - #   2215              - #  2220                         - - Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gl - #n Glu Met Gly Gly Asn      2225                2230 - #                2235 - #               2240        - - Ile Thr Arg Val Glu Ser Glu Asn Lys Val Va - #l Ile Leu Asp Ser Phe                      2245 - #               2250  - #              2255             - - Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Gl - #u Ile Ser Val Pro Ala                  2260     - #           2265      - #          2270                 - - Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gl - #n Ala Leu Pro Val Trp              2275         - #       2280          - #      2285                     - - Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Gl - #u Thr Trp Lys Lys Pro          2290             - #   2295              - #  2300                         - - Asp Tyr Glu Pro Pro Val Val His Gly Cys Pr - #o Leu Pro Pro Pro Lys      2305                2310 - #                2315 - #               2320        - - Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Ar - #g Thr Val Val Leu Thr                      2325 - #               2330  - #              2335             - - Glu Ser Thr Leu Ser Thr Ala Leu Ala Glu Le - #u Ala Xaa Arg Ser Phe                  2340     - #           2345      - #          2350                 - - Gly Ser Ser Ser Thr Ser Gly Ile Thr Gly As - #p Asn Thr Thr Thr Ser              2355         - #       2360          - #      2365                     - - Ser Glu Pro Ala Pro Ser Gly Cys Pro Pro As - #p Ser Asp Ala Glu Ser          2370             - #   2375              - #  2380                         - - Xaa Xaa Ser Met Pro Pro Leu Glu Gly Glu Pr - #o Gly Asp Pro Asp Leu      2385                2390 - #                2395 - #               2400        - - Ser Asp Gly Ser Trp Ser Thr Val Ser Ser Gl - #u Ala Asn Ala Glu Asp                      2405 - #               2410  - #              2415             - - Val Val Cys Cys Ser Met Ser Tyr Ser Trp Th - #r Gly Ala Leu Val Thr                  2420     - #           2425      - #          2430                 - - Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Il - #e Asn Ala Leu Ser Asn              2435         - #       2440          - #      2445                     - - Ser Leu Leu Arg His His Asn Leu Val Tyr Se - #r Thr Thr Ser Arg Ser          2450             - #   2455              - #  2460                         - - Ala Cys Gln Arg Gln Lys Lys Val Thr Phe As - #p Arg Leu Gln Val Leu      2465                2470 - #                2475 - #               2480        - - Asp Ser His Tyr Gln Asp Val Leu Lys Glu Va - #l Lys Ala Ala Ala Ser                      2485 - #               2490  - #              2495             - - Lys Val Lys Ala Asn Xaa Leu Ser Val Glu Gl - #u Ala Cys Ser Leu Thr                  2500     - #           2505      - #          2510                 - - Pro Pro His Ser Ala Lys Ser Lys Phe Gly Ty - #r Gly Ala Lys Asp Val              2515         - #       2520          - #      2525                     - - Arg Cys His Ala Arg Lys Ala Val Thr His Il - #e Asn Ser Val Trp Lys          2530             - #   2535              - #  2540                         - - Asp Leu Leu Glu Asp Asn Val Thr Pro Ile As - #p Thr Thr Ile Met Ala      2545                2550 - #                2555 - #               2560        - - Lys Asn Glu Val Phe Cys Val Gln Pro Glu Ly - #s Gly Gly Arg Lys Pro                      2565 - #               2570  - #              2575             - - Ala Arg Leu Ile Val Phe Pro Asp Leu Gly Va - #l Arg Val Cys Glu Lys                  2580     - #           2585      - #          2590                 - - Met Ala Leu Tyr Asp Val Val Thr Lys Leu Pr - #o Leu Ala Val Met Gly              2595         - #       2600          - #      2605                     - - Ser Ser Tyr Gly Phe Gln Tyr Ser Pro Gly Gl - #n Arg Val Glu Phe Leu          2610             - #   2615              - #  2620                         - - Val Gln Ala Trp Lys Ser Lys Lys Thr Pro Me - #t Gly Phe Ser Tyr Asp      2625                2630 - #                2635 - #               2640        - - Thr Arg Cys Phe Asp Ser Thr Val Thr Glu Se - #r Asp Ile Arg Thr Glu                      2645 - #               2650  - #              2655             - - Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pr - #o Gln Ala Arg Val Ala                  2660     - #           2665      - #          2670                 - - Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gl - #y Gly Pro Leu Thr Asn              2675         - #       2680          - #      2685                     - - Ser Xaa Gly Glu Asn Cys Gly Tyr Arg Arg Cy - #s Arg Ala Ser Gly Val          2690             - #   2695              - #  2700                         - - Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cy - #s Tyr Ile Lys Ala Arg      2705                2710 - #                2715 - #               2720        - - Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cy - #s Thr Met Leu Val Cys                      2725 - #               2730  - #              2735             - - Gly Asp Asp Leu Val Val Ile Cys Glu Ser Al - #a Gly Val Gln Glu Asp                  2740     - #           2745      - #          2750                 - - Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Me - #t Thr Arg Tyr Ser Ala              2755         - #       2760          - #      2765                     - - Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr As - #p Leu Glu Leu Ile Thr          2770             - #   2775              - #  2780                         - - Ser Cys Ser Ser Asn Val Ser Val Ala His As - #p Gly Ala Gly Lys Arg      2785                2790 - #                2795 - #               2800        - - Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pr - #o Leu Ala Arg Ala Ala                      2805 - #               2810  - #              2815             - - Trp Glu Thr Ala Arg His Thr Pro Val Asn Se - #r Trp Leu Gly Asn Ile                  2820     - #           2825      - #          2830                 - - Ile Met Phe Ala Pro Thr Leu Trp Ala Arg Me - #t Ile Leu Met Thr His              2835         - #       2840          - #      2845                     - - Phe Phe Ser Val Leu Ile Ala Arg Asp Gln Le - #u Glu Gln Ala Leu Asp          2850             - #   2855              - #  2860                         - - Cys Glu Ile Tyr Gly Ala Cys Tyr Ser Ile Gl - #u Pro Leu Asp Leu Pro      2865                2870 - #                2875 - #               2880        - - Pro Ile Ile Gln Arg Leu His Gly Leu Ser Al - #a Phe Ser Leu His Ser                      2885 - #               2890  - #              2895             - - Tyr Ser Pro Gly Glu Ile Asn Arg Val Ala Al - #a Cys Leu Arg Lys Leu                  2900     - #           2905      - #          2910                 - - Gly Val Pro Pro Leu Arg Ala Trp Xaa His Ar - #g Ala Arg Ser Val Arg              2915         - #       2920          - #      2925                     - - Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala Al - #a Ile Cys Gly Lys Tyr          2930             - #   2935              - #  2940                         - - Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Ly - #s                          2945                2950 - #                2955                             __________________________________________________________________________

What is claimed:
 1. A method for producing an hepatitis C virus (HCV)qlycoprotein having mannose-terminated glycosylation, wherein less thanabout 10% of the total N-linked carbohydrate on said HCV glycoprotein issialic acid, and further wherein said HCV qlycoprotein is selected fromthe group consisting of a glycoprotein expressed from the E1 region ofHCV and a glycoprotein expressed from the E2 region of HCV comprisingthe steps of:growing a mammalian host cell transformed with a structuralgene encoding an HCV glycoprotein expressed from either the E1 region ofHCV or the E2 region of HCV in a suitable culture medium; causingexpression of said structural gene under conditions inhibitingsialylation; and isolating said HCV qlycoprotein from cell culture bycontacting said HCV glycoprotein with a mannose-binding protein specificfor mannose-terminated glycoproteins.
 2. The method of claim 1, whereinsaid conditions inhibiting sialylation comprise expression of theqlycoprotein at a rate sufficient to inhibit transport of glycoproteinsfrom the endoplasmic reticulum to the golgi.
 3. The method according toclaim 1, wherein said conditions inhibiting sialylation comprise asufficient amount of a calcium modulator to cause release of proteinswithin the host cell's endoplasmic reticulum.
 4. The method according toany of claims 1-3, wherein said qlycoprotein is expressed from the E1region of HCV.
 5. The method according to any of claims 1-3, whereinsaid glycoprotein is expressed from the E2 region of HCV.
 6. The methodaccording to any of claims 1-3, wherein said glycoprotein is an E1/E2aggregate.
 7. The method according to any of claims 1-3, wherein saidmannose-binding protein is a lectin selected from the group consistingof ConA and GNA.
 8. The method according to any of claims 1-3, whereinsaid mannose-binding protein is immobilized on a support.
 9. The methodaccording to claim 8, wherein said contacting comprises incubation ofsaid expression product in a column comprising a mannose-binding lectinimmobilized on a support, for a period of at least one hour; and whereinsaid isolating comprises eluting said glycoprotein with mannose.
 10. Themethod of any one of claims 1-3, wherein said glycoprotein is an E1/E1aggregate.
 11. The method of any one of claims 1-3, wherein saidglycoprotein is an E1/E2 aggregate.